Device and bioanalytical method utilizing asymmetric biofunctionalized membrane

ABSTRACT

Bioanalytical device that includes a biofunctional component and an optional sensor component. The device includes arrays of addressable, durable, asymmetric biofunctional membranes containing protein transducers capable of unidirectional transport of analytes. Suitable protein transducers include members of the ATP-binding cassette family, such as P-glycoprotein.

This application claims the benefit of U.S. Provisional Application Ser.No. 60/326,862, filed 3 Oct. 2001, which is incorporated herein byreference in its entirety.

BACKGROUND OF THE INVENTION

The design and synthesis of bioinspired materials such as syntheticlight harvesting complexes, artificial ion channels, artificial muscles,has captured the imagination of many individual research groups. Veryfew efforts, however, have been directed toward the full integration ofbiologic and synthetic materials for the creation of hybridbiofunctional devices. Accomplishments in the area ofbacteriorhodopsin-based optoelectronic devices (Birge, IEEE Comput. 25,56 (1992)), gramicidin-based biosensors (Cornell et al., Nature 387,580-583 (1997)), and photochemical triad-driven ATP synthase processes(Steinberg-Yfrach et al., Nature 392, 479-482 (1998)) constituteimportant advances that have involved multidisciplinary investigativegroups to direct the design, synthesis, processing, structural analysis,and performance testing of these devices.

It has been 20 years since the first demonstration thatLangmuir-Blodgett films could be used to assemble lipid bilayers onsolid surfaces (Von Tschamer et al., Biophys. J. 36(2), 421-427 (1981)).This initial work inspired intense interest in biotechnologies based onsupported lipid bilayers because of the important role that membraneproteins play in living systems. Unfortunately, applications ofsupported lipid membranes have been limited by their instability. Recentbreakthroughs by Cornell et al (Nature, 1997, 387:580) and Bieri et al.(Nature Biotechnol., 1999, 17:1105) in anchoring chemistries and proteinorientation, respectively, have produced durable asymmetricbiofunctional membranes Channel proteins are embedded in the membranesto provide biofunctionality. Cornell et al. describe the incorporationof gramicidin dimers in membranes formed from a tethered lipid bilayer.Bieri et al. describe the use of conventional bilayers with theG-protein coupled receptor, bacteriorhodopsin. This protein is orientedwith respect to the surface using a streptavidin-biotin interaction.Others have used a silyl-modified polyethylene glycol (PEG) to tether asupported membrane to glass.

Despite these advances, the stability of planar membrane structuresremains an issue. Moreover, conventional methods are limited to the useof channel proteins and require chemical modification of the proteinthat is often non-specific or difficult to control. Biofunctionalmembranes exhibiting increased stability and/or broader utility areneeded in order to meet the complex needs of nanotechnology andbiotechnology.

SUMMARY OF THE INVENTION

Arrays of addressable, durable, asymmetric biofunctional membranesaccording to the present invention are capable of unidirectionaltransport of analytes such as chemical and biological agents, therebyenabling a number of new technologies including functional screeningtechnologies of membrane proteins and their interaction with compoundsof interest; chemical and biological agent detection anddecontamination; bioprocess separations; and environmentally-responsivematrices (e.g., surgically-implanted drug reservoirs, dermal patches andsubcutaneous implants) for drug delivery under feedback control. Theactive transport structures in these biofunctional devices are orientedin large arrays and supported to provide transport directionality andmechanical stability.

Accordingly, one aspect of the invention is directed to a bioanalyticaldevice that includes a biofunctional component and, optionally, a sensorcomponent. The biofunctional component includes, as components, amembrane film, a protein transducer directionally oriented within themembrane film, and a support substrate. The membrane film has an outersurface and an inner surface, such that the inner surface is in contactwith an aqueous compartment that is disposed between the membrane filmand the support substrate. The inner surface of the membrane is thus influid communication with the support substrate. The aqueous compartmentpreferably contains a detection moiety for detecting the activity of theprotein transducer. Preferably, the activity of the protein transduceris detectable by detecting ATP hydrolysis.

Optionally, the biofunctional component of the bioanalytical device alsoincludes a transducer orienting layer disposed between the membrane filmand the support substrate. The transducer orienting layer may includeone or more orienting moieties that interacts with the proteintransducer to orient the protein within the membrane film. Alternativelyor in addition, orienting moieties may be included on or within thesupport substrate. The transducer orienting layer may also include adetection moiety for detecting the activity of the protein transducer.

The membrane film can be formed from synthetic or naturally occurringcomponents. It can take the form of a bilayer or a monolayer. Exemplarycomponents of the membrane film include one or more lipids, bolalipids,bolaamphiphiles, triblock copolymers and/or a hydrogels.

The protein transducer can be, for example, a multi-drug resistanceprotein, a multi-drug resistance-associated proteins ormitoxantrone-resistance protein. A preferred protein transducer is aprotein which is a member of the ATP-binding cassette superfamily, suchas P-glycoprotein. Gramicidin is another preferred protein, and isespecially useful in an embodiment of the device wherein the membranefilm is a monolayer, such as that formed by a bolalipid orbolaamphiphile. In a monolayer film, the gramicidin can advantageouslyfunction as a monomer.

The support substrate can be formed from, for example, a nanoporousmaterial or a solid. Examples of suitable materials include silicon,gold and γ-alumina. The device optionally includes a gold electrodehaving an orienting moiety attached thereto, such that the orientingmolecule interacts with the protein transducer to orient the proteinwithin the membrane film.

The optional sensor component of the bioanalytical device detects asignal generated upon operation of the biofunctional component. Thesignal is indicative of the activity of the protein transducer. Thissignal can be, for example, a chemical signal, an optical signal, anelectrochemical signal, an electrical signal, and/or an electromagneticsignal.

The bioanalytical device is typically organized as an array ofbiofunctionalized membranes in fluid communication with the supportsubstrate, the biofunctionalized membranes comprising the membrane filmand the protein transducer directionally oriented with the membranefilm. Advantageously, the biofunctional component of the bioanalyticaldevice can be fabricated as a replaceable cartridge.

In another aspect, the invention includes a method for making thebioanalytical device. A biofunctional component can be fabricated byapplying a hydrogel precursor solution to the surface of a supportsubstrate; applying a gellating agent to the hydrogel precursor to causegellation of the hydrogel precursors to yield a transducer orientinglayer in physical contact with the surface of the support substrate; andapplying a protein transducer and a membrane film material to thetransducer orienting layer to yield a membrane film in fluidcommunication with the transducer orienting layer. The inner surface ofthe membrane film defines an aqueous compartment between the transducerorienting layer and the membrane film. Optionally, the transducerorienting layer is integrated into the support substrate.

In another aspect, the invention includes a method for analyzing ananalyte, such as a drug, using a bioanalytical device as describedherein. In one embodiment, the analyte is contacted with the outersurface of the membrane film of a bioanalytical device as describedherein, such that the analyte passes into or across the membrane film.The activity of the protein transducer is then detected, whereinactivity of the protein transducer is indicative of the efflux of theanalyte from the outer surface of the membrane film. In an alternativeembodiment, protein transducers having uptake activity rather thenefflux activity can be used to analyze an analyte. Uptake of the analyteinto the aqueous compartment is then detected.

In yet another aspect, the invention includes a method for identifyingan inhibitor of a protein transducer using a bioanalytical device asdescribed herein. In one embodiment, the outer surface of a membranefilm is contacted with (a) an analyte capable of being activelytransported by the membrane-embedded protein transducer and (b) acandidate inhibitor of the protein transducer. It is then determinedwhether the active transport of the analyte by the membrane-embeddedprotein transducer is inhibited by the candidate inhibitor compared tothe active transport of the analyte by the membrane-embedded proteintransducer in the absence of the candidate inhibitor. In anotherembodiment, the method can be used to identify an inhibitor of a proteintransducer having uptake activity instead of efflux activity.

In yet another aspect, the invention includes a kit for making abioanalytical device which includes, as components, membrane filmmaterial; hydrogel precursors; support substrate; and packaging andinstructions for use of the bioanalytical device for analysis of theactivity of a membrane-embedded protein transducer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cross-sectional view of a supported biofunctionalizedmembrane.

FIG. 2 is a two-dimensional hypothetical model of human P-glycoprotein(P-gp) structure based on hydropathy plot analysis of primary amino acidsequence. The ATP binding/utilization domains are circled with theWalker A, B and “linker dodecapeptide” or “signature sequence” (LSGGQ)motifs are designated by the letters “A”, “B” and “C.” Putativeglycosylation sites are represented by squiggly lines. Serine residuesknown to be phosphorylated are shown as dark circles with an attachedand encircled “P”. The filled-in circles show many of the positions ofmutations that change substrate specificity in human P-gp.

FIG. 3 is a schematic representation of (A) gramicidin in a bilayermembrane in the “on” state; (B) gramicidin in a bilayer membrane in the“off” state; and (C) gramicidin in a bolalipid monolayer membrane.

FIG. 4 is a schematic representation of different supported membraneconfigurations: (A) PEG chains attached to both the support and themembrane; (b) PEG chains only attached to the solid support; and (c) PEGchains grafted to one material, either the support or the membrane.

FIG. 5 is a schematic representation of three elements used to constructthe microreactor arrays.

FIG. 6 shows the structures of naturally occurring bolalipids isolatedfrom thermophilic archaebacteria.

FIG. 7 shows representative synthetic bolalipids that form stabilizedsupported membranes.

FIG. 8 shows the delamination process for bilayer membranes.

FIG. 9 shows a comparison of drops of 50 wt % glycerol in water producedby using different wave forms.

FIG. 10 shows impedance behavior of Au electrodes during sequentialdeposition onto Au electrodes: bare Au electrodes; addition of SSC20BAS(2); addition of C20BAS (2+1); and addition of gramicidin(2+1+gramicidin), wherein reference numbers 1 and 2 refer to the lipidsin FIG. 7.

FIG. 11 shows solvent resistance of supported membranes on Au electrodes(A) SSC20BAS-DPPC; (B) SSC20BAS-C20BAS-GA.

FIG. 12 shows capacitance and single channel measurements (A)capacitance of C20 bolalipid; (B) bolalipid C20:gramacidin-D (100:1ratio).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention provides a platform for the creation of arrays ofaddressable, durable asymmetric biofunctional membranes that are capableof unidirectional transport of analytes such as chemical and biologicalagents. Very large arrays, e.g., arrays of about 10⁵ supportedbiofunctionalized membranes are feasible. These arrays facilitatefunctional screening of membrane proteins and their interaction withlibraries of the compounds of interest.

This platform forms the basis for a unique bioanalytical instrument thatis capable of directly determining the activity of potentialchemotherapy compounds with protein transducers such as P-glycoproteinat high throughput rates. This instrument permits screening of new andexisting libraries of chemotherapy compounds for their biologicalactivity without using expensive and indirect cell based assays. Thisinstrument also facilitates the development of a fundamentally newapproach to the characterization of membrane protein behavior. Thecentral role that these proteins play in all aspects of cellular biology(e.g., cell signaling, cell motility, energy conversion, proteinexpression, cell division) cannot be over-emphasized. Further, thebiofunctionalized membranes potentially have other biotechnologicalapplications including: bioprocess separation;environmentally-responsive matrices (dermal patches, subcutaneousimplants, etc) for drug delivery under feedback control; chemical andbiological agent detection; and hazardous agent decontamination.

The bioanalytical device of the invention includes a biofunctionalcomponent and, optionally, a sensor component. The biofunctionalcomponent includes a membrane film, a protein transducer directionallyoriented within the membrane film, and a support substrate in fluidcommunication with the membrane film and defining an aqueous compartmentbetween the membrane film and the support substrate. Optionally butpreferably, the biofunctional component also includes a transducerorienting layer disposed between the membrane film and the supportsubstrate. In a variety of alternative embodiments, one or more of theconstituent components of the biofunctional component can be integratedso as to form a composite structure. For example, the biofunctionalizedmembrane film can be integrated within the support, thereby increasingmembrane stability and facilitating membrane processing.

FIG. 1 presents a cross-sectional view of one illustrative embodiment ofthe biofunctional component of the device. Protein transducer 2, in thiscase P-gp, is embedded in membrane film 4 to yield biofunctionalizedmembrane 6. Bolalipids 8 are used to form the membrane film 4. Proteintransducer 2 contains a His₆ tag 10 which allows tethering of theprotein transducer 2 to a transducer orienting layer 12 using apolyethylene glycol (PEG) tether 14. Transducer orienting layer 12,which can be, for example, a hydrogel, contains a chemically responsivematerial, such as an environmentally sensitive dye. Inner aqueouscompartment 16 is disposed between biofunctionalized membrane 6 andtransducer orienting layer 12 and serves as a reservoir for substrate(in this embodiment, ATP) that drives the pumping action of proteintransducer 2. Support substrate 18, which can include a gold, silicon orγ-alumina surface, for example, and is in this case formed from silicon,provides mechanical support for biofunctionalized membrane 6 and otherdevice components and can also serve orienting and/or sensing functions.The mechanism pumping is depicted as a series of events (i.e. within thebiofunctionalized membrane, moving from left to right) that produces anet analyte 20 flux from the inner aqueous compartment 16 to outeraqueous phase 22.

In a preferred embodiment, the protein transducer is P-glycoprotein(P-gp) and P-gp-mediated active drug transport is detected as a changein pH or ionic strength due to the coupled hydrolysis of ATP. Sensorsdeveloped from these asymmetric structures provide a direct indicationof a drug candidate's susceptibility for P-gp pumping across themembrane, which will serve as a powerful tool for screening druglibraries for those leads that are likely to remain inside the targetcell type, as discussed in more detail below. Discovery of novelcompounds from high-throughput screening according to the presentinvention may lead to improved chemotherapeutic agents for refractorytumors.

The sensor component of the device, if present, detects a signalgenerated upon operation of the biofunctional component. The signal canbe a chemical signal, an optical signal, an electrochemical signal, anelectrical signal, an electromagnetic signal, or any other type ofdetectable signal. Typically, a signal generated by the device afterapplication of a chemical reagent for analysis is indicative of theactivity of the protein transducer. In embodiments containing both thebiofunctional component and the sensor component, the biofunctionalcomponent is optionally but conveniently supplied as a replaceablecartridge, while the sensor component remains a permanent part of thedevice. It should be noted, however, that there are applications such asbioseparations wherein a sensing modality is not necessary.

The invention also includes a kit for making the biofunctional componentof the bioanalytical device. The kit contains membrane film material,hydrogel precursors and a support substrate. Alternatively, the kitcontains a preformed support substrate/transducer orienting layer,together with the membrane film material. Also included is packaging andinstructions for use of the bioanalytical device for analysis of theactivity of a membrane-embedded protein transducer. The kit allows theuser to construct a membrane film embedded with a custom proteintransducer of the user's own choosing.

Membrane Film

The membrane film can be formed from one or more natural or syntheticmaterials, such as lipids or other hydrophobic materials.Archaebacterial lipids from halophilic bacteria and Methanogen (i.e.,any of the various archaebacteria (see Archaea) that produce methane;they include such genera as Methanobacillus and Methanothrix), andbacterial bolalipids from thermophilic bacteria are particularly useful.Other materials that can be used for the biomembrane film include, forexample, non-fouling template-polymerized bilayers, bolaamphiphiles,triblock copolymers (such as PEG-PiB-PEG and PEO-PPO-PEO) and hydrogels.The membrane film is preferably planar. See, e.g., Salafsky et al.,Biochemistry, 1996, 35, 14773-14781; Raguse et al., Langmuir, 1998, 14,648-659; Groves et al., Langmuir, 2001, 17, 5129-5133; and Groves etal., Biophys. J., 1996, 71, 2716-2723; and U.S. Pat. No. 6,228,326,Boxer et al. for examples of supported membrane films. The use ofsynthetic lipids or lipid-like molecules allows the incorporation ofstructural modifications that can increase the stability of thesupported membrane.

The membrane film can take the form of a bilayer, such as a lipidbilayer, or it can take the form of a monolayer structure. Naturallyoccurring lipids, with polar head groups and hydrophobic tails,typically form a bilayer. Boxer and coworkers have shown that supportedmembrane film arrays can be formed by liposome fusion with microcontactprinted surfaces. Analysis of these films has revealed that the membranebilayers retain their fluidity via entrapment of a nm-sized aqueousphase between the membrane coating and solid support. Proteoliposomefusion with solid supports has further shown that proteoliposome canfuse with acid-treated glass surfaces to give supported membranes withthe membrane proteins oriented in a vectorial fashion. However, afeature common to supported bilayer membranes is their tendency todelaminate from the solid support within 24 hours.

Accordingly, a monolayer structure is preferred for the membrane film ofthe biofunctional component of the device. A monolayer structure can beformed from, for example, bolalipids, bolaamphiphiles, or amphiphilictri-block copolymers. Supported bolalipid membranes are especiallypreferred. Optionally, lipid components of the membrane can be tetheredto the support substrate (e.g., Cornell et al., Nature, 1997,387:580-583.)

Biofunctionalization of the Membrane Film

Embedded within the membrane film are directionally orientedmembrane-spanning proteins that function as molecular transporters(“protein transducers”), yielding a “biofunctionalized membrane.” Thebiofunctionalized membrane can be considered a biomimetic structure. Itfunctions as an active transport layer, such that analytes can beactively transported across it via the protein transducers.

Protein Transducers

The invention is not limited to the use of any particular proteintransducer, any membrane protein that can function as a moleculartransporter across the membrane film can be used in thebiofunctionalized membrane. It should be noted that the bioanalyticaldevice is equally suitable in applications involving efflux of ananalyte or uptake of an analyte, depending on the bioactivity of theprotein transducer selected. Examples of protein transducers than can beused in the present technology include proteins associated withmulti-drug resistance such as the product of the human MDR1 gene,P-glycoprotein (including MDR efflux pump, peptide efflux pump andphospholipid flippase), and the product of the human BSEP gene, the bilesalt export pump (both members of the APT-binding cassette superfamily,described below) and other multi-drug resistance-associated proteins(MRPs), mitoxantrone-resistance proteins (MXR1/BCRP/ABCP/ABSG2), andporins. Another example is cyt bc, a complex of cytochrome b and c. ATPsynthase can be driven by the H⁺ gradient generated by co-immobilizedcyt bc complex.

ABC transporters. The ATP-binding cassette (ABC) superfamily containsboth uptake and efflux transport systems. ATP hydrolysis, typicallywithout protein phosphorylation, energizes transport. There are dozensof families within the ABC superfamily, and family generally correlateswith substrate specificity. The transporters of the ABC superfamilyconsist of two integral membrane domains/proteins and two cytoplasmicdomains/proteins. The uptake systems (but not the efflux systems)additionally possess extracytoplasmic solute-binding receptors. Both theintegral membrane channel constituent(s) and the cytoplasmicATP-hydrolyzing constituent(s) may be present as homodimers orheterodimers.

The superfamily includes prokaryotic ABC-type uptake transporterfamilies including, without limitation: Carbohydrate UptakeTransporter-1 (CUT1); Carbohydrate Uptake Transporter-2 (CUT2); PolarAmino Acid Uptake Transporter (PAAT); Hydrophobic Amino Acid UptakeTransporter (HAAT); Peptide/Opine/Nickel Uptake Transporter (PepT);Sulfate Uptake Transporter (SulT); Phosphate Uptake Transporter (PhoT);Molybdate Uptake Transporter (MolT); Phosphonate Uptake Transporter(PhnT); Ferric Iron Uptake Transporter (FeT);Polyamine/Opine/Phosphonate Uptake Transporter (POPT); Quaternary AmineUptake Transporter (QAT); Vitamin B₁₂ Uptake Transporter (VB₁₂T);Chelate Uptake Transporter (FeCT); Manganese/Zinc/iron Chelate UptakeTransporter (MZT); Nitrate/Nitrite/Cyanate Uptake Transporter (NitT);Taurine Uptake Transporter (TauT); Putative Cobalt Uptake Transporter(CoT); Thiamin Uptake Transporter (ThiT); and Brachyspira IronTransporter (BIT).

The superfamily includes bacterial ABC-type efflux transporter familiesincluding, without limitation: Capsular Polysaccharide Exporter (CPSE);Lipooligosaccharide Exporter (LOSE); Lipopolysaccharide Exporter (LPSE);Teichoic Acid Exporter (TAE); Drug Exporter-1 (DrugE1); Putative Lipid AExporter (LipidE); Putative Heme Exporter (HemeE); β-Glucan Exporter(GlucanE); Protein-1 Exporter (Prot1E); Protein-2 Exporter (Prot2E);Peptide-1 Exporter (Pep1E); Peptide-2 Exporter (Pep2E); Peptide-3Exporter (Pep3E); Probable Glycolipid Exporter (DevE); Na⁺ Exporter(NatE); Microcin B17 Exporter (McbE); Drug Exporter-2 (DrugE2); MicrocinJ25 Exporter (McjD); Drug/Siderophore Exporter-3 (DrugE3); Putative DrugResistance ATPase-1 (DrugRA1); and Putative Drug Resistance ATPase-2(DrugRA2).

The superfamily also includes other ABC-type efflux transporterfamilies, mostly eukaryotic, including, without limitation: MultidrugResistance Exporter (MDR) (includes P-glycoprotein); Cystic FibrosisTransmembrane Conductance Exporter (CFTR); Peroxysomal Fatty Acyl CoATransporter (FAT); Eye Pigment Precursor Transporter (EPP); PleiotropicDrug Resistance (PDR); α-Factor Sex Pheromone Exporter (Ste); ConjugateTransporter-1 (CT1); Conjugate Transporter-2 (CT2); MHC PeptideTransporter (TAP); Heavy Metal Transporter (HMT);Cholesterol/Phospholipid/Retinal (CPR) Flippase; and Mitochondrial Fe/SProtein Exporter (MPE).

Human ATP-Binding cassette transporters number about 48 (seehttp://nutrigene.4t.com/humanabc.htm) and include the ABC1 family(subfamily ABCA), MDR family (subfamily ABCB), MPR family (subfamilyABCC), ALD family (subfamily ABCD), OABP family (subfamily ABCE) GCN20family (subfamily ABCF) and White family (subfamily ABCG). For a reviewyeast ABC transporters, see Taglicht et al., Meth. Enzymol. 1998,292:130-162.

P-glycoprotein. The successful treatment of metastatic and disseminatedcancer is to a large degree dependent upon the effectiveness ofcytotoxic anticancer drugs. Some commonly used chemotherapeutic agentsare the Vinca alkaloids, the anthracyclines, the epipodophyllotoxins,taxol, and actinomycin D. Although these natural product compounds sharelittle to none of the same chemistry, they all are amphipathic moleculeswith planar aromatic rings that preferentially partition into organicsolvents. Unfortunately, most cancers either are intrinsically resistantto any initial treatment with these therapeutic compounds or acquireresistance to a broad spectrum of these agents over time (Gottesman etal., Annu. Rev. Biochem. 62, 385 (1993)).

It is well established that this broad-based resistance results, inlarge part but not solely, from the overexpression of a 170 kDa plasmamembrane polypeptide known as the multidrug transporter orP-glycoprotein (P-gp), encoded by the multidrug resistance MDR1 gene inhumans (Gottesman et al., Annu. Rev. Genet. 29, 607 (1995)). P-gp is amember of the ATP binding cassette (ABC) superfamily of membranetransporters, with toxin binding domains localized within thetransmembrane regions. It is an energy-dependent multidrug transporterthat reduces the accumulation of an extremely broad range ofstructurally unrelated hydrophobic and amphipathic molecules withincells. P-gp is known to efflux cytotoxic drugs out of cells and limitthe influx of drugs into cells. Known substrates include vinblastine,daunomycin, actinomycin D, taxol, colchicine, verapamil and rapamycin.

P-gp is believed to play a protective barrier role in normal tissues,defending them from the damaging effects of toxins, dietary drugs andother harmful environmental agents. However, because it plays a majorrole in drug resistance, P-gp is making it the subject of intenseinterest to the pharmaceutical community. Many different human cancersexpress the MDR1 gene at levels sufficient to confer multidrugresistance. Based on an analysis of several hundred different humancancers, it can be estimated that approximately 50% of human cancerswill express the MDR1 gene at some time during therapy (Ambudkar et al.,Annu. Rev. Pharmacol. Toxicol. 39, 361 (1999)). The World HealthOrganization has also estimated that multidrug resistant bacteriaaccount for near 60% of all hospital-acquired infections. The majortherapeutic challenge of multidrug resistance in cancer and infectiousdisease clearly illustrates why there are large drug discovery effortsat major pharmaceutical companies that are directed toward this problem.Thus, although there are many membrane proteins of interest,P-glycoprotein (P-gp) is preferred for use in the biofunctionalizedmembranes of the invention due to the important role it is thought toplay in broad-based resistance to chemotherapies.

Human P-gp is an integral membrane protein comprised of two homologoushalves each thought to span the plasma membrane bilayer six times witheach half containing an ATP binding site (Gottesman et al., Annu. Rev.Genet. 29, 607 (1995)). (FIG. 2). Topology studies reveal that both theamino and carboxyl termini of P-gp are located in the interior of thecell. The drug binding sites are localized to the transmembrane domainsof the transporter (Greenberger, J. Biol. Chem. 268, 11417 (1993); andBruggemann et al., J. Biol. Chem. 267, 21020 (1992)) whereas the ATPsites are cytosolic. Hydrolysis of adenosine triphosphate (ATP) on thecytosolic surface of the membrane is coupled to the transport ofsubstrate molecules out of the cell, which means that active transportis coupled to energy consumption and local pH.

ATP binding and hydrolysis are essential for the proper functioning ofP-glycoprotein, including drug transport (Horio et al., Proc. Natl.Acad. Sci. USA 85, 3580 (1988)), however, how the energy of ATPhydrolysis is transduced to transport this large variety of hydrophobicagents out of the plasma membrane or the cytoplasm into theextracellular milieu remains unknown. One proposed model is that P-gpmay reduce intracellular drug concentrations by acting as a “hydrophobicvacuum cleaner” effectively increasing drug efflux and decreasing druginflux by the recognition and removal of compounds from the membranebefore they reach the cytosol to elicit their cytotoxic effects (Ravivet al., J. Biol. Chem. 265, 3975 (1990)). The major sites of interactionof several P-gp inhibitors and substrates have been localized totransmembrane regions 5, 6, 11 and 12 as determined by photoaffinitylabeling, digestion with proteases or cyanogen bromide, and specificimmunoprecipitation with antibodies directed against polypeptideepitopes of P-gp. Additionally, one of the most useful and informativeexperimental approaches used to probe substrate specificity has involvedthe study of mutant and chimeric molecules that are either naturallyoccurring or artificially engineered. Many of these mutations in humanP-gp molecule affect changes in the drug specificity of the transporterrelative to the wild-type molecule. These mutations are found scatteredthroughout the molecule but are generally localized to the transmembraneregions with most of the functionally relevant mutations found intransmembrane domains 5, 6, 11 and 12.

Because multidrug resistance has proven to be an enormous clinicalproblem, there is great interest in both academic and industriallaboratories in the development of novel and clinically usefultherapeutic agents to serve as P-gp substrates and inhibitors. Thesecompounds could be used to facilitate the treatment of human cancers aswell as serve as diagnostic agents for the non-invasive evaluation ofP-gp expression in certain tumors. With the advent of combinatorialchemistry methodologies, large numbers of these compounds can begenerated. At present, however, there are no rapid ways to screen largenumbers of these compounds for activity. Existing vesicle and cell-basedassays for analyzing human P-gp activity are slow and difficult to adaptto high throughput methods. There is a clear need for rapid, highthroughput methods to screen combinatorial libraries of P-gpchemosensitizers and substrates.

The present invention provides medium and high-throughput methods forscreening potential new P-gp substrates and modulators. The invention isexemplified by arrays of human P-glycoprotein (P-gp)-functionalizedasymmetric membranes. Arrays constructed from the biofunctionalizedmembrane structures can be used to identify promising pharmaceuticalcompounds and fingerprint unknown environmental toxins based on P-gpactivity patterns.

Specific domains of P-gp involved in substrate recognition have recentlybeen identified; mutational studies further indicate that it will bepossible to engineer designer transporters that would selectivelydiscriminate between molecular species. The present invention envisionsa broad range of active transport membrane structures that areconstructed based on the same operating principles, but differing intheir molecular transport specificity.

A particularly promising application of P-gp is as a transducing elementfor energy coupled transport in biofunctionalized membrane constructs.Genetically engineered designer transporters that recognize specifiedclasses of substrate molecules can be realized in sensor arraysconstructed from such materials. These devices are useful in a widevariety of applications, including: first-line rapid screening of largedrug libraries in high throughput fashion, characterization of thepumping mechanism of P-gp, or fingerprinting unknown environmentaltoxins and functionally-related classes of pharmacological agents basedon P-gp activity patterns.

Gramicidin. Gramicidins are known to increase the permeability ofbiological membranes to protons and alkali-metal cations by formingtransient dimeric ionophoric channels through the membrane, and areuseful as protein transducers in the present invention. In a bilayer,gramicidin cycles between “on” and “off” states as the monomers movewithin the fluid membrane (FIGS. 3A and B). Alternatively, in thebiofunctional membrane of the present invention, gramicidin can placedbe in a perpetually “on” state by embedding it into a monolayer membrane(FIG. 3C). In a monolayer, only a monomer of gramicidin is needed toallow passage of analytes through the membrane.

Orientation of the Protein Transducers within the BiofunctionalizedMembrane

The invention is not limited by the method used to orient the proteintransducers within the membrane film. Many orientation strategies relyon the concept of “tethering” the protein to a component of thetransducer orienting layer, if present, or directly to a component ofthe support substrate.

Methods for orienting the protein transducers in the membrane film rangefrom the very specific to the relatively nonspecific. Specificligand/receptor interactions such as biotin-streptavidin can beutilized, or the proteins can incorporate His-, Myc-, FLAG- orHA-affinity tags. A polyhistidine-Ni²⁺-NTA (or IDA) binding interactionis especially useful. The proteins can be engineered to contain epitopesor antigens that bind an antibody present in the transducer orientinglayer or as part of the support substrate. Orientation can be achievedby taking advantage of ligand binding sites (e.g., ATP binding sites) orsites of metal chelation either present in the naturally occurringprotein or engineered into it. Protein-, carbohydrate- or lipid-nucleicacid chimeras can be used, thereby allowing the formation of DNAduplexes via hybridization with complementary nucleic acids presentwithin the transducer orienting layer or on the surface of aDNA-modified support substrate. Covalent reactions with thiols or aminesare another option for orienting the proteins within the membrane layer.As a less specific method, charge clusters can be used to orient theprotein transducer using electrostatic interactions, such aselectrostatic adsorption onto a charged surface. Oligonucleotide-lipidconjugates, DNA aptamers, inclusion complexes with cyclodextrinmonolayers, and/or microcontact-printed thiols on hydrogel supports, arefurther examples of methods that can be used to fix the orientation ofthe protein transducers and provide mechanical support.

In embodiments of the device that include a transducer orienting layer,it is typically composed primarily a polymeric layer such as a hydrogellayer, a sol-hydrogel layer or composite sol-hydrogel layer.Functionalities important for tethering the protein transducers can beintegrated into the polymeric layer either covalently or noncovalently.The hydrogel is typically formed from cross-linked polymers. However,the invention is not limited by the nature of the materials used to makethe transducer orienting layer.

The technique used to “tether” the protein transducer is preferably onethat allows the requisite amount of membrane fluidity. Fluidity isimportant so that the proteins can move within the membrane. Fluiditycan be achieved in a number of different ways. The interaction betweenthe protein and the tethering component in the transducer orientinglayer or the support substrate may be strong, yet reversible. Forexample, the transducer orienting layer can take the form of acyclodextrin film, and the protein transducer (derivatized or engineeredto contain a hydrophobic inclusion ligand at the end of the tether) canreversibly bind the cyclodextrin to form an inclusion layer.Alternatively, the interaction the protein and the tethering componentmay be covalent and essentially irreversible, but the tetheringcomponent is able to move within the transducer orienting layer, as in ahydrogel layer, a sol-hydrogel layer or composite sol-hydrogel layer.

There are at least three possible ways to design the polymerictransducer orienting layer, such as a PEG layer, to avoid direct contactbetween the biofunctionalized membrane and the underlying supportsubstrate (FIG. 4). In FIG. 4A, the PEG molecules are chemically boundto both the support and the membrane. FIG. 4B depicts the case where PEGis chemically bound to the support, but not the lipid membrane. Thepolymer layer repels the membrane, leading to a supported membrane ontop of the extended PEG layer. The third case, FIG. 4C, corresponds tothe situation where PEG units are grafted single surfaces, either thelipid membrane or membrane support. Theoretical calculations shows thatwhen PEG is chemically bound to both the membrane and support, a muchsharper energy minimum is observed for these interactions, suggestingthat this approach may be a preferred way to support thebiofunctionalized membrane at the energetically-favorable distance ofchoice.

It may be desirable to provide additional room between the membrane andsupport for effective function of the protein transducer. This can beachieved by using a softer interaction. For example, for some polymersattaching polymers of half chain length to each surface theoreticallyresults in interactions that are similar in magnitude as the case withonly single point attachments.

The transducer orienting layer can contain molecules having detectormoieties, such as dye molecules to detect, for example, ATP hydrolysis,which is indicative of molecular transport through the membrane embeddedprotein transducer; it can also contain molecules having orientingmoieties such as polymers that function to orient the membrane-embeddedprotein transducers within the membrane film.

Support Substrate

The support substrate provides mechanical support for thebiofunctionalized membrane. It is not limited by the type of materialused for fabrication. For example, in one embodiment the supportsubstrate can be a nanoporous structure; in other embodiments it can bea solid. Suitable materials for use in fabricating the support substrateinclude anodic γ-alumina or nanoporous silica structures. It can alsotake the form of a glass, gold (Au), an Au electrode, or an indium tinoxide (ITO) electrode.

The transducer orienting layer and support substrate can be distinctlayers, or they can be integrated. In some embodiments, a single layercomposed of, for example, cross-linked polymers with dyes and orientingmolecules covalently attached, can function as both the support and thetransducer orienting layer.

Additional Structural Considerations

If desired, the distance between the biofunctionalized membrane and thehydrogel/support substrate support layer(s) can be controlled by the useof water soluble polymers, such as oligo(ethylene glycol) spacer units,that extend between the biofunctionalized membrane and the transducerorienting layer/support substrate, tethering them together and actingsomewhat like the springs of a mattress underlying the biofunctionalizedmembrane. X-ray reflectivity and x-ray standing wave measurements can beused to precisely determine this distance.

In an alternative embodiment, the transducer orienting layer/supportsubstrate layer(s) are replaced by a planar gold electrode, and thetransport of charged analytes is detected electrochemically.

Microfabrication Technologies for the Support Substrate and TransducerOrienting Layer

The biofunctional membranes are oriented in the devices and supported toprovide transport directionality and mechanical stability. Support isprovided by a substrate, and a transducer orienting layer, such as asol-gel matrix, is optionally added to the surface of the supportsubstrate to assist in orienting the membrane proteins and provideoptically addressable groups for the transduction of local changes inthe chemical environment.

In recent years, there has been a merger of microelectronics andbiological sciences to develop “biochip” devices. The term biochip hasbeen used in various contexts, but can be generally defined as amicro-fabricated device that is used for processing (delivery,processing, and analysis) of biological species (molecules, cells,etc.). Asymmetric supported P-gp membrane arrays can be formed on thesurface of 100×100 micron reaction wells that will allow rapid screeningof up to 100,000 distinct combinations of membrane protein-drugcandidate. These arrays can be assembled by microfabricating reactionchambers from Si (100) wafers using standard bulk micromachiningtechniques. Surface chemistries can then be used to orient P-gp and thehost lipid membrane in the reaction chambers.

The device can be conveniently assembled in stages. Initially, forexample, a hydrogel precursor solution can be added via ink jet head tothe support. Then a gellating agent can be added, again via ink jethead, to cause gellation of the hydrogel precursors. To this is addedthe protein transducer (for example, as a detergent micelle solution),followed by the membrane film material (as a vesicle solution that alsocontains ATP as a solute) to yield the biofunctionalized structure. Asnoted herein, the transducer orienting layer can be overlaid on thesupport substrate, or integrated within it.

The use of a support allows both the outer surface (akin to theextracellular surface) or inner surface (akin to the cytosolic surface)of the biofunctionalized membrane to be addressed (FIG. 1). The innersurface is in contact with the aqueous compartment that contains theentrapped aqueous solution. The outer surface of the membrane can beaddressed by addition of reagents that potentially activate P-gpfunction; the inner surface can be addressed—albeit slowly—by samplingthe solution residing within the hydrogel support. For example, if ATPis hydrolyzed, its reaction products will slowly infuse throughout thewater-filled voids of the hydrogel. Since ATP and its hydrolysisproducts are confined within a compartment bounded by the membrane filmand the container walls, these agents have no other option but todiffuse away from the membrane. Sampling of this aqueous environment andanalysis by mass spectrometry, scintillation, and the like can then beused to detect the extent of ATP hydrolysis. Fabrication of thebiofunctionalized structures on planar substrates utilizes commonmembrane protein purification techniques, such as decylglucosidemicellar solubilization combined with surface binding and orientationusing molecular recognition elements such as histidine- or myc-tags (seebelow).

Existing nanoporous materials (e.g. anodic γ-alumina) represent suitablehosts. Second-generation materials, such as self-assembled nanoporoussilica structures (e.g., SBA-15) created using templates formed by theself-assembly of amphiphilic triblock copolymers (Yang et al., Science282, 2244-2246 (1998)) are also envisioned to allow further integrationof the biofunctionalized membrane into the nanoporous support toincrease membrane stability and facilitate membrane processing. In apreferred embodiment, nanoporous alumina membranes are used to supportthe biofunctional membranes. The nanoporous membranes are ideally suitedfor incorporation in the devices because they have highly uniform poresof size 20-100 nm, a high elastic modulus, and can withstand hightemperatures. Standard wafer bonding techniques can be used to bond thesilicon wafers to the membrane. After the silicon-nanoporous membrane isassembled, inkjet printing can be used to deliver the sol-gel precursorsto the active surface of the membrane, followed by photochemical orthermal gellation of the material.

The transducer orienting layer can serve one or more of the followingfunctions: 1) as a mechanical support matrix, 2) as a scaffold fordisplaying specific affinity elements that promotes proteinimmobilization within the biofunctionalized membrane such that thecytoplasmically accessible ABC sites (i.e. the ATP hydrolysis domains)are oriented toward the substrate, 3) as a matrix within whichpH-sensitive or ion-specific dyes can be immobilized (by modifying thehydrogel with commercially-available, isothiocyanate forms of the dye)to enable rapid, highly sensitive optical detection of the environmentnear the ABC sites, and 4) when structurally integrated with thesubstrate or serving dual functions as transducer orienting layer andsubstrate, as a nanoporous substrate that allows reagent access to bothsurfaces of the protein-embedded membrane, a level of control that noother membrane-based technique can offer.

Microreaction chambers can also be constructed from bulk micromachinedstructures and nanoporous membranes. Asymmetric biofunctional membranescan be provided on the micron scale in arrays of at least 100,000distinct combinations of membrane protein-drug candidate compound.Microreaction chambers (eg., 10×10 μm² reaction chambers) can befabricated with sol-gel—substrate supports at a density of 100,000chambers/cm². Formation of distinctly addressable arrays is based on theability to dispense 10⁻¹⁵ liter volumes using inkjet dispensingtechnologies (see below), and construct reaction chambers in which thesefluids can be dispensed on durable biofunctionalized membranes that havebeen assembled on nanoporous supports.

The reaction chambers can be assembled by microfabricating grid likestructures from silicon and fusing them with the nanoporous support.FIG. 5 shows how the two grids and nanoporous support can be assembled.The reaction chambers themselves can be machined from a Si (100) waferusing standard bulk micromachining techniques (Petersen, Proc. IEEE 70,420-457 (1982)). These layers serve to localize the solutions are bedelivered through microfluidic means. Surface chemistries, e.g., silaneor thiol monolayers, are used to control adsorption of proteins andlipids in the reaction chambers. Protein and membrane chemistries thatare compatible with the “ink jet” technologies are preferred for use inconstructing the addressable, oriented arrays of the invention.

Design and synthesis of membrane materials for the biofunctionalizedmembrane. Two competing physical chemical issues impact the function andruggedness of the biofunctionalized membrane-based device—membranefluidity and mechanical stability. Membrane fluidity is required forfunction of membrane proteins. The membrane materials used forimmobilization of the membrane proteins are therefore preferably intheir liquid crystalline phase at the operating temperature of thedevice (i.e. pumping activity may be lost if the host membrane materialis in the gel phase).

Even though the membrane fluidity can be controlled experimentally bycontrolling temperature, it is undesirable to use this approach, sincethe thermal stability of physiologically active proteins such as P-gpmay be limited above physiological temperatures (37° C.). The lowtemperature limit of the proposed device, which contains aqueous reagentcompartments, is near 0° C. This narrow temperature range for deviceoperation places stringent demands on the structure and composition ofthe host membrane material.

Preferred materials are naturally occurring or synthetic lipid compoundsthat have the ability to form stable membranes with low intrinsicpermeabilities toward the substrate, for example ATP, a substrate forP-gp. Synthetic materials can be used as dopants within the membranesformed by natural materials. In addition, since the functional devicesundergo multiple reagent and analyte addition steps, the membranematerial also needs to be physically robust.

It is known that supported membrane film arrays can be formed by fusionof liposomes with microcontact printed surfaces. Analysis of these filmshas revealed that the membrane bilayers retain their fluidity viaentrapment of a nm-sized aqueous phase between the membrane coating andsolid support. Proteoliposome fusion with solid supports has also beenshown to yield supported membranes with photosynthetic reaction centersoriented in a vectorial fashion. Most of the known methods forstabilizing membranes involve polymerization of vinylic lipid monomerswithin the bilayer. Unfortunately, in the past this approach has alwaysled to loss of membrane protein activity, either through loss ofmembrane fluidity or via direct protein-monomer reactions.

The apparently mutually-incompatible material demands of membranefluidity and mechanical stability can be resolved by, for example, usingbolalipid materials such as bolaamphiphiles that are tethered to anunderlying polymeric scaffold. Bolalipids are a family of natural andsynthetic bipolar membrane lipids that are patterned after the membranematerials found in the extremely thermophilic organisms such asSutfolobus acidocaldarius, that thrive under conditions as harsh as pH 1and ≧100° C. S. acidocaldarius cell membranes consist of componentsderived from unique glyceryl tetraether lipids often bearing two polarheadgroups and isoprenyl-based or cyclopentane ring-modified membranespanning alkyl chains that connect the two headgroups together, andacyclic and macrocyclic structures (FIG. 6). Representative syntheticbolalipids are shown in FIG. 7.

Bolalipids preferentially form aqueous dispersions with very littlecurvature of the membrane due to their centrosymmetric molecularstructures (e.g. 1 and 3 in FIG. 7) and preferential placement of onepolar headgroup at each interface of the bolamembrane (i.e. anelongated, rather than U-shaped, conformation is the predominantorientation). Recent pulsed field gradient NMR experiments indicate thatthe diffusion coefficient of these lipids is more than two orders ofmagnitude slower than the lateral diffusion rate of conventionalmonopolar lipids. This observation, taken together with thecross-fracturing behavior seen in freeze-fracture TEM and the highlyordered Raman spectra of these membranes, is consistent with theoccurrence of an elongated, transmembrane conformation of these lipids.This is expected to significant impact the stability of supportedmembranes formed from these materials.

The unusual stability of these lipids is attributed to their resistancetoward hydrolysis (due to the chemically robust ether linkage) anddelamination (due to the presence of membrane-spanning alkyl chains, achemical architecture that effectively “crosslinks” the membrane in adirection normal to the membrane surface). These features have beenincorporated within a series of bolalipids containing C₁₆- and C₂₀-basedtransmembrane chains that have been synthesized and found to possessexceptional membrane stability relative to conventional bilayer membranematerials (Patwardhan et al., Org. Lett. 1, 241 1999; DiMeglio et al.,Langrnuir 16, 128 (2000); and Patwardhan et al., Langmuir 16, 10340(2000)).

The incorporation of these transmembrane chains into devices can be usedto prevent the delamination processes that plague conventional supportedbilayer membrane films (FIG. 8). Homologous bolalipids bearing C₂₈- andC₃₂-transmembrane chains, for example, are compatible with the membranedomain dimensions of P-gp and are synthesized using the reaction pathwaydescribed by Thompson and coworkers (Patwardhari et al., Org. Lett. 1,241 (1999)), except that the transmembrane chain contains methylbranching sites and/or olefinic residues to prevent the close packing ofalkyl chains that promotes gellation (solidification) of the membrane. Avariety of physical techniques, e.g., differential scanning calorimetry,electron spin resonance, and fluorescence recovery after photobleaching,can be used to identify lipids that retain a liquid crystalline statewithin the 0° C.-37° C. operating temperature range of the device.

For further stability, the biofunctionalized membrane can be attached toa extramembranous polymeric scaffold disposed within the aqueouscompartment, as described in Example III. Two different approaches, theuse of a molecular spring, e.g., a polyethylene glycol (PEG) “mattress,”and adsorption onto a porous hydrogel support, can be utilized.

Amphiphilic molecules and dye-modified macromolecules can be used toorient membrane proteins such as P-gp at the surface of the transducerorienting layer and assemble them within durable membrane films. Forexample, C-terminal histidine-tagged variants of human P-gp can beexpressed and purified by metal affinity chromatography. Thehistidine-tagged proteins interact with Ni²⁺ and(α-(α-lysinenitrilotriacetic acid) (NTA), a property which can beexploited in soft immobilization techniques for protein orientation andimmobilization within the membrane films. Orienting molecules, such asbifunctional polyethylene glycols of varying molecular weights that havebeen modified with α-(α-lysinenitrilotriacetic acid) (NTA) and ω-thiolsubstituents, can be disposed within or synthesized into the transducerorienting layer. An important factor in the synthesis ofnitrilotriacetic acid conjugates is the use of methyl ester protectedintermediates until their hydrolysis in the final deprotection step.This approach enables the use of conventional synthetic methodologythroughout the pathway and avoids the tedious and difficult separationsthat would result if free carboxylate intermediates were used instead.

Alternatively, gold electrodes can be used in place of thehydrogel/nanoporous support. The proteins can be immobilized onto thegold electrodes with their drug binding domains oriented away from theelectrode surface using bifunctional polyethylene glycols of varyingmolecular weights that have been modified withα-(α-lysinenitrilotriacetic acid) and co-thiol substituents as describedabove. Thiol adsorption of these derivatives onto Au orients the NTAligand toward the aqueous phase where both Ni²⁺ and micellar his-tagP-gp can bind. Addition of bolalipids to these partially coatedelectrodes, followed by dilution above the critical micellarconcentration (CMC), induces self-assembly of planar bolalipid membranescontaining highly oriented P-gp. Biotin-streptavidin recognition hasbeen similarly used to produce bacteriorhodopsin supported bilayerfilms.

Impedance spectroscopy (IS) can be used to detect ion flux acrosssupported bolalipid membrane films. This technology thus advantageouslymakes use of a new sensor based on either optical or impedancespectroscopic analysis of supported membranes that could greatlyaccelerate the discovery of new P-gp reversing agents and substrates.

Impedance spectroscopy is capable of providing the sensitivity,simplicity, and speed necessary for reliable analysis. For example, ifit is assumed that the reaction chambers are 100 μm×100 μm and 10 nmdeep (i.e., the minimum membrane-support separation), the volume of thereaction chamber would be 1×10⁻¹¹ cm³. If the starting pH is 7, thenthere would be 602H⁺ ions within that volume in the absence of othersources of ions. If we further assume that the starting resistivity ofthe solution within the chamber is 0.1 Ω-cm, then the resistance of sucha chamber would be about 1MΩ, which is measurable. The key question,however, concerns the magnitude of the change in resistivity due to agiven pH change. For simple electrolytes, this change is approximatelylinear. Thus, if we have a reaction chamber that initially contains600H⁺ ions that give a 1MΩ resistance, then the addition of another 60H⁺ions due to P-gp activity (i.e., a 10% increase) should produceapproximately a 100 kΩ change in resistance which is easily measurable.If it assumed that an ATP-driven transducer protein, such as P-gp, ispresent at a surface density of 1 pump/μm² and that it is capable ofreleasing 1 H⁺/sec due to ATP hydrolysis, then a 100 μm×100 μm reactionchamber would have total of 10⁴ pumps, producing a large increase in H⁺concentration. It is expected that changes in resistivity arising fromP-gp catalyzed ATP hydrolysis will be readily measurable using impedancemethods.

Microfluidics. There are currently three techniques for deliveringfluids to suitable substrates, e.g. chips, for creating high-densitymicro-arrays (Schena et al., TIBTECH 16, 301-306 (1998)). They arephotolithography, contact printing with pin tools, and ink jet printing.These techniques can be contrasted based on their technical as well ascost benefits or drawbacks.

A primary advantage of photolithography, e.g. as developed and practicedby Affymetrix of Santa Clara, Calif., over other techniques is that itis capable of achieving extremely high-density arrays. Theoretically,the technique is capable of creating spot sizes of 10 μ², i.e. 10microns by 10 microns, or smaller. However, the technique is expensivedue to the costs associated with creating photomasks.

Contact printing with pin tools, a technique popularized by PatrickBrown of Stanford University, CA, is, by contrast, a relativelyinexpensive method for creating arrays. Its drawbacks include theslowness of the arraying process, the requisite contact of the printingtool with the substrate for transfer of the working fluid(s) from thepin to the substrate and the accompanying problem of contamination, andthe relative coarseness of the arrays that the method can achieve. Thismethod is unlikely to achieve spot sizes smaller than 100 μ² (Rose, Bio.Techniques Publishing (2000)).

Inkjet technology allows the production of highly reproducible dropscomposed of mixtures of one or more components, and ink jet printing isemerging as the most versatile and promising of all arraying techniques.First, it is both a non-contact and a low-cost method of arraying inwhich the spots are created by ejecting tiny drops from one or(typically) many nozzles onto the substrate. Second, while the currentof state-of-the art with ink jet printing is spot sizes of roughly 100μ², the technique is capable of creating arrays approaching theresolution possible with photolithography. Ink jet printing can becarried out in either the continuous (CIJ) or the drop-on-demand (DOD)modes (Le, J. Imaging Sci Technol. 42, 49-62 (1998)). Furthermore, theDOD mode can be carried out using either piezo or thermal dropgeneration methods. To date, virtually all researchers have adoptedpiezo DOD printing in arraying applications. Regardless of which mode ofink jet printing is used, the method is also extremely fast because asingle nozzle of an ink jet arraying system can eject several thousanddrops per second.

While ink jet printing has been around for more than 30 years, itsscientific underpinnings have only started to be understood recently(Wilkes et al., Phys. Fluids 11, 3577-3598 (1999); and Notz et al.,Phys. Fluids 13, 549-552 (2001)). Drop formation from an ink jet nozzleentails both small length and time scales. Typical nozzle radii R are onthe order of tens of microns or less, typical drop sizes R_(d) areroughly the same as R, and typical drop formation times are on the orderof a few or tens of microseconds. Once the drops are formed, they travelthrough the air and impact the substrate. The drops either successfullydeposit on the substrate or else bounce off and/or shatter upon impact.Following successful deposition on the substrate, the drops spread to afinal spreading radius or spot size R_(s) that exceeds R_(d). Thedeposition and spreading processes also last on the order of a few ortens of microseconds. Given these scales and the so-called free boundarynature of the drop formation and deposition processes, it is a challengeto describe theoretically and to monitor experimentally the creation ofarrays.

The objectives of this task are to eject small drops from one or morepiezo DOD ink jet nozzles, deposit them on a suitable substrate surface,and ensure the quality of the ejection and deposition processes. Currentink jet drop dispensers are limited in that the radii of drops that theygenerate are roughly the same as the radii of the nozzles that producethem. We have recently developed a novel method for generating dropswith radii much smaller than those of nozzles that produce them (Chen etal., Phys. Fluids. (2001)), as shown in FIG. 9. This new method can beimplemented to reduce drop size and hence the spot size that can beachieved with ink jet printing. Since the drop liquids may contain lipidparticulates and proteins, they are complex non-Newtonian liquids. Thus,successful jetting and deposition of such liquids requires that theirsurface and bulk rheologies be well characterized (Yildirim et al.,Chem. Eng. Sci. 56, 211-233 (2001)). Surface characterization tools areused to evaluate solution parameters such as shear and extensionalviscosities.

Adsorption of microdrops. Deposition of 10-100 μm diameter microdropletsonto substrate surfaces using ink jet nozzles requires a detailedunderstanding of the surface properties of both the solid and solutionsurfaces. The droplets may contain lipid particulates such asmultilamellar liposomes, unilamellar vesicles, and micellar solutions ofmembrane proteins. Since lipid molecules are surface active (adsorbstrongly) at both air/water and solid/water interfaces, a portion ofthem adsorbs onto those surfaces. For the air/water interface, the mainconsequence is a change in the dynamic surface tension, which affectsthe drop formation dynamics. For the solid/water interface, the mainconsequence is the change in the wettability of the solid membrane. Forboth interfaces, adsorption implies a material loss, which becomes moreacute the larger the surface-to-volume ratio and the lower theconcentration. Furthermore, protein adsorption on hydrophobic solidsurfaces or on water may cause unfolding or denaturation. Hence,interactions of lipids and proteins with interfaces must be controlled,and should be understood at fundamental level.

Adsorption and surface tension of bolalipids and membrane proteins suchas P-gp at the air/water and the solid/water interfaces can be evaluatedusing direct methods such as ellipsometry and at the air/waterinterface, and similarly at the solid/water interface. The solid/surfacecan be probed from the liquid side with IRRAS, or from the solid side(where possible) using total internal, reflection IR (or ATR-FTIR).Which component or components adsorb first or ultimately (atequilibrium) can be determined by analyzing dynamically the surfacecomposition. The use of “sacrificial” adsorbates, which maypreferentially adsorb and thereby protect the biofunctional bolalipidand P-gp materials, is also envisioned.

Optical sensing techniques and characterization. Read-out of theactivity of specific protein-reagent pairs can be achieved using pHsensitive dyes immobilized in the transducer orienting layer. Opticaltechniques (such as total internal reflection fluorescence (TIRF)microscopy, infrared reflection-adsorption spectroscopy (IRRAS), andellipsometry) and atomic force microscopy (AFM) techniques can be usedto characterize the chemical and physical state of structures containingthe biofunctionalized membranes. Immunochemistry techniques can also beused to probe the structure of the films and compare with models of thefilm at various protein surface loadings. The transport activity of theprotein-embedded films (i.e., the biofunctionalized membranes) may becompared with its activity in vesicles (control). Conventional bulksilicon micromachining techniques can be used to fabricate microreactorarrays to which the nanoporous supports are bonded. Inkjet printing,microcontact printing, and/or UV-lithography, for example, can be usedassemble the transducer orienting layers and biofunctional asymmetricmembranes, and also to introduce the agents to be studied.

Operation of the Device

In operation, the chemical agent to be tested or analyzed (such as adrug, a toxin, or any other naturally occurring or synthetic molecule ormolecular complex) is applied to the device via ink jet deposition tothe outer or “extracellular” side of the membrane film, and makes itsway through the membrane film via diffusion or some other mechanism.Once across the membrane into the aqueous compartment (the “cytosolic”region), the agent potentially induces the activity of the proteintransducer such that it is pumped out of the cytosolic region. Theactivity of the protein transducer can be detected in any number ofways, as illustrated above. In a preferred embodiment, the activity ofthe protein transducer is detected via the hydrolysis of ATP present inthe aqueous compartment.

Operation of one embodiment of the device is illustrated in FIG. 1. Theprotein transducer, P-gp, is oriented in the membrane film by virtue ofthe interactions between a His₆ tag on the protein and a polyethyleneglycol linker in the transducer orienting layer as described in ExampleIII. An analyte is delivered to the outer side of the biofunctionalmembrane. The analyte moves into and/or across the membrane, binds toP-gp and is pumped out in process driven by ATP hydrolysis. The analyteis effluxed from the outer or (“extracellular”) side of the device, andthe pump is reactivated.

EXAMPLES

The present invention is illustrated by the following examples. It is tobe understood that the particular examples, materials, amounts, andprocedures are to be interpreted broadly in accordance with the scopeand spirit of the invention as set forth herein.

Example I Validation of Sensor Performance Using Gramicidin (Ga)

Cyclic voltammetry was used as a probe of supported bolalipid membraneformation on Au electrodes. Immersion of the electrodes into ethanolicsolutions of 2 at 25° C. for various times produced surfaces withvoltammetric responses that displayed decreasing electrochemicalreversibility of Fe(CN)₆ ³⁻ as 2 deposition increased. (Note that thereference numbers 1 and 2 used in this example refer to bolalipidstructures 1 and 2 as shown in FIG. 7.) These data suggest that longerimmersion times produced films that were increasingly capable ofblocking Fe(CN)₆ ³⁻/Fe(CN)₆ ⁴⁻ access to the Au surface, such that after3 hours, the 2 anchor lipid films were deposited as an essentiallycontinuous layer that passivated the surface toward ferri/ferrocyanideredox processes. Time-dependent impedance and phase angle properties of2-deposited gold electrodes showed similar effects of increasingimpedances with increasing deposition time. The experiments describedbelow utilized 60 minutes immersion times for 2 solutions prior to 1 or1-GA supported membrane depositions.

The effect of GA surface density on supported membrane impedance wasdetermined using electrodes that had been coated with supportedbolalipids by pretreatment with 2 prior to immersion for 24 hours intosonicated 1-GA vesicles containing varying amounts of GA. The 1-GAvesicle dispersions were prepared by sonicating dry 1-GA films in water(nominal GA:bolalipid molar ratios ranging from 1:500-1:50; 2 mM totallipid concentration). Impedance measurements with these 1-GAsupportedmembrane electrodes showed impedance decreases with increasing GAconcentration over the entire 1-10³ Hz range, indicating that GA isincorporated within the 1 bolalipid membrane as a functional ionchannel. The relatively small changes in impedance observed as thegramicidin:bolalipid ratio was increased by an order of magnitudesuggests that ion flux through the channel must be sufficiently fastthat increased surface densities of gramicidin channels has littleinfluence on impedance behavior. This observation is consistent with²³Na NMR ion flux measurements made previously in our laboratory forgramicidin-containing 1 vesicles.

The effects of gramicidin loading on supported bolalipid membraneelectrode impedance was further investigated in a sequential depositionexperiment (FIG. 10). Adsorption of 2 onto Au electrodes for 1 hour togive partial surface coverage produced significant increases inimpedance. Immersion of these pretreated electrodes into bolalipidvesicle solutions where gramicidin was either absent or present (i.e. 1and 1-GA vesicles, respectively) produced membrane coated electrodesthat had substantially different impedance characteristics in 10⁻¹-10²Hz frequency range. As the data in FIG. 11 show, immersion of the2-treated electrode into 2 mM 1 membrane solutions for 24 hours furtherincreased the sensor impedance, due to vesicle-surface fusion withformation of a continuous, impermeable 1 membrane with high-impedance.When the 2 electrodes were immersed in 1-GA membrane solutions for 24hours (1:100 molar ratio GA:1), however, the observed impedances werelower—approaching that of bare Au—than for either the 2 or 2-1 supportedbolalipid membrane coatings. These data further support the involvementof GA as an ion transporter in these supported membrane constructs. Wealso infer from these results that the 1 hour pretreatment of gold withethanolic solutions of 2 produces films that are disordered anddiscontinuous (i.e. containing defect sites due to low surface coveragesand/or poor bolalipid packing). These disordered surfaces becomewell-ordered upon adsorption and fusion of bolalipid vesicle dispersionsonto the partially-covered electrode surfaces. When 1 vesicles are used(i.e. gramicidin-free bolalipid vesicles), higher impedances areobserved due to the annealing of surface defects by the more highlyordered 1 membrane. When 1-GA vesicles are used, the highly ordered 1monolayer membranes nonetheless produce Au electrode surfaces with lowerimpedance due to the activity of monomeric GA channels that are activelytransporting cations across the supported membrane films.

Capacitance experiments (FIG. 12) also showed that C20 bolalipidmembranes, without gramicidin, exhibited no ion flux, whereas additionof gramicidin to the monolayer resulted in ion flux across the membrane.

Gold electrodes bearing supported bolalipid membranes are alsoremarkably rugged compared to conventional supported bilayer membranes.Cyclic voltammetry experiments reveal that sequential washings withwater, ethanol, and methylene chloride had relatively little effect onthe voltammetric response of membrane-treated Au electrodes (FIG. 11).This contrasts with the behavior of DPPC-based supported membraneelectrodes prepared in a similar manner, which displayed far lessresilience toward identical solvent treatments that the bolalipidmembrane surfaces. These films are presumably more robust because themembranes are more highly-ordered than the DPPC films where hydrophobicmismatch between the bilayer and SSC20BAS monolayer lipids createssolvent-accessible defect sites.

Example II Hydrogel Layer/Support Structure

Monomers or polymers are loaded into the micropores of the solid supportmaterial using ink-jet methods. The hydrogel support inside themicropores can be prepared by two methods: crosslinking polymerizationof monomers and crosslinking reactions of water-soluble polymers. Sinceit is desirable to have the chemically active functional moieties on thepolymer backbone, chemically active monomers or polymers are preferredThe following monomers and polymers are good candidates for makinghydrogels.

-   -   1. Monomers that can react with the hydroxyl groups of proteins        or polysaccharides (e.g. acryloyl chloride and glycidyl        acrylate)    -   2. Monomers that can react with amine groups of proteins and        polysaccharides (e.g. methacryloyl chloride,        acryloxyethylisocyanate, allyl glycidyl ether, and acrolein)    -   3. Monomers that can react with carboxyl groups of proteins or        polysaccharides (e.g. vinyl acetate)        The above monomers are used in conjunction with other monomers        that are chemically inert. They are acrylamide,        vinylpyrrolidone, and hydroxyethyl methacrylate, for example.        The chemically active groups of the functional monomers are used        to covalently graft P-gp proteins to the hydrogel surface. Once        P-gp is grafted to the hydrogel surface, the remaining        functional groups are quenched by using suitable small molecular        weight quenchers, such as amino acids.        Polymers to form hydrogels. Water-soluble polymers that can        react with bifunctional crosslinking agents can be used to form        chemical gels. Examples include:    -   1. Polymers containing hydroxyl groups (e.g. dextran, starch)    -    These polymers can be crosslinked with bis(epoxy propyl) ether,        divinyl sulfone, carbonyldiimidazole, epichlorohydrin, and        glutaraldehyde.    -   2. Polymers containing amine groups.    -    These polymers can be crosslinked with bisimidate,        bis(sulfonsuccinimidyl) suberate, bis(p-nitrophenyl)adipate,        bisepoxide, and glutaraldehyde.    -   3. Polymers containing carboxyl groups.

Polyepoxides have been used frequently to crosslink this type ofpolymer. Water-soluble polymers can also be made into physical gelsusing non-covalent crosslinks. Physical gels can be formed by ionicassociations, hydrophobic interactions, and stereocomplex formation, forexample. The most commonly used method in biomedical and pharmaceuticalareas is the calcium ion-induced gelation of sodium alginate.

Any monomer and/or polymer described above can be used for:

1. Crosslinking polymerization. Monomers (e.g., acryloyl chloride,glycidyl acrylate, or acrolein) are mixed with acrylamide orvinylpyrrolidone with the molar concentration of chemically activemonomers ranging from 1 mol % to 30 mol %. A crosslinking agent, such asbisacrylamide, is added and its concentration is varied from 1 mol % to10 mol % of monomers. Azobisisobutyronitrile is used as the initiator atthe concentration of 1 mol % of monomers. Once the hydrogel is formed,the chemically active groups are used for covalent grafting of proteinsto the surface of the formed hydrogels. Once the grafting is complete,the remaining functional groups are deactivated by adding excessglycine.2. Physical gelation. Sodium alginate is mixed with ATP beforeintroduction into the micropores. The concentration of sodium alginatewill be maintained at about 1 w/v %. From our experience, the viscosityof alginate in this concentration range is high enough for using ink jetdispensers. At the same time, the concentration is also high enough toform physical gels in the presence of calcium ions. Once sodium alginateis filled in the pores, the bottom layer of the solid support will beimmersed into a calcium ion solution so that the gelation begins fromthe surface. Once the gel is formed, the carboxyl groups of alginatemolecules are used for chemical grafting of proteins to the hydrogelsurface. If the increase in the thickness of the hydrogel layer isnecessary, the alginate gel can be exposed to poly(L-lysine) solution(0.1 w/v %) to coat the hydrogel surface with polycation. Then, theamine groups of the poly(L-lysine) can be used for further chemicalmodification, or additional alginate can be immobilized on top of thepolycations. This process can be repeated as needed to increase thedevice durability.

One of the advantages physical gelation over crosslinking polymerizationis that the gelation process is very simple and, at the same time, easyto control the regions between the polymer chains that influence thediffusion rate of molecules inside the hydrogel. The concentrations ofsodium alginate and calcium ions, as well as the time of calcium iondiffusion into the alginate gel, can be adjusted to obtain physical gelsof the desirable properties.

Example III P-glycoprotein Isolation and Formation of theBiofunctionalized Membrane

P-gp can be tagged with 6-10 histidine residues at the C-terminuswithout affecting function. Wild-type P-gp containing a six histidinetag at the C-terminus (P-gp-H₆) was purified from insect cells usingmetal affinity chromatography. P-gp-H₆ has been shown to be 85% pure asindicated by silver staining. Using the rapid dilution method toreconstitute the protein into proteoliposomes, Vi-sensitive drugstimulated ATPase activity of P-gp-H₆ was determined. The reconstitutedprotein demonstrated high specific activity (5.8 μmol/min/mg protein) inthe presence of 30 μM verapamil. This rapid dilution methodreconstitutes approximately 20% of the starting material, 50% of whichis catalytically active, as determined by permeabilization ofproteoliposomal membranes with alamethicin. Therefore, afterreconstitution, only 10% of the input protein is accessible for themeasurement of ATPase activity. This yield of functional P-gp was usedto calculate the specific activity of the protein.

Vanadate (V_(i))-sensitive ATPase activity can be measured inproteoliposomes reconstituted with purified P-gp-H using the followingassay. Purified protein (0.35-0.45 μg/mL) containing 1.25%octylglucoside and 0.1% lipid mixture is rapidly diluted 20-fold toreconstitute P-gp into proteoliposomes in a 13×100 mm glass test tube ina reaction mixture containing 50 mM Tris-HCl (pH 6.8), 125 mM KCl, 5 mMMgCl₂, and 1 mM dithiothreitol (DTT) in the presence or absence of 300μM sodium orthovanadate and incubated at 37° C. for 3 minutes.Subsequently, 1 μL of dimethyl sulfoxide (DMSO) or 3 mM verapamil(prepared in DMSO) is added and the reaction mixture incubated for anadditional 3 min at 37° C. The reactions are started by the addition of2.5 mM ATP and incubated at 37° C. for 20 minutes. The reactions arestopped by the addition of an equal volume of 5% (v:v) sodium dodecylsulfate (SDS) and the amount of phosphate released determinedspectrophotometrically.

The protein is immobilized onto a nanoporous/hydrogel membrane supportwith the drug binding domains oriented away from the support. This canbe accomplished using bifunctional polyethylene glycols (Sackmann,Science 271, 43 (1996); and Wong et al., Biophys. J. 77, 1445 (1999)) ofvarying MW's that have been modified with α-(α-lysinenitrilotriaceticacid) (NTA) and co-thiol substituents. Reaction of the thiol substituentwith the underlying support layer orients the NTA ligand toward theaqueous phase where both Ni²⁺ and micellar his-tag P-gp can bind.

When electrochemical detection methods are preferred in the specificapplication, his-tagged membrane proteins are added to gold electrodesthat are partially coated with the same bifunctional polyethyleneglycols bearing α-(α-lysinenitrilotriacetic acid) (NTA) and ω-thiolsubstituents as described above. Dilution of the oriented membraneproteins below the CMC of the protein-solubilizing detergent by additionof a large excess of bolalipid vesicles is expected to induceself-assembly of planar bolalipid membrane films containing highlyoriented P-gp. Biotin-streptavidin recognition has been similarly usedto produce bacteriorhodopsin supported bilayer films (Bieri et al.,Nature Biotechnology 17, 1105-1108 (1999)). The resultingbiofunctionalized membranes are now configured to detect membraneprotein-mediated events via their effect on impedance at the goldelectrode surface.

Monoclonal antibodies directed against the histidine tag can be used asconfirmation of orientation. The orientation of P-gp in these supportedmembrane structures can be further established by differentialrecognition of the protein with antibodies specific for distinct siteson the protein. Three monoclonal antibodies that recognize humanP-glycoprotein have been developed. MRK-16 is specific for an externalepitope in the first extracellular loop of human P-gp whereas C219(Kartner et al., Nature 316, 820 (1985)) recognizes regions within theinternal cytoplasmic nucleotide binding domains. UIC2, aconformationally-sensitive antibody whose reactivity is increased in thepresence of P-gp transport substrates, ATP-depleting agents, ormutations that reduce the level of nucleotide binding by P-gp, is alsospecific for the external portion of the protein. Use of theseantibodies is expected to allow for the recognition of either side ofP-gp after reconstitution of the protein into the supported membraneenvironment and aid in determining the functional state of the protein.

Alternatively, other epitope tags—including myc and FLAG tags—can begenetically engineered into the C-terminus and the first extracellularloop of P-gp. Insertions and limited deletions of these regions havebeen shown to have little to no effect on P-gp function. The addition ofthese epitope tags to the N-terminus and the effect of the insertion onfunction is evaluated. Orientation of these proteins in the supportedmembrane can be assessed by antibody recognition using commerciallyavailable antibodies.

If P-gp activity is compromised because the trapped water compartment issmall and prevents coupling of the two ATP binding sites, different MWPEGs can be used as flexible spacers. If these undergo delamination, themechanical rigidity of the support can be increased using dextran films.

If P-gp activity is affected by membrane viscosity and/or specific lipidcomponents within the biofunctionalized membrane, bolalipids containingan increasing density of methyl branching sites at the membrane core canbe used. These bolalipids represent a family of membrane materials whosephase transition temperatures (Tm) are near 15° C. The biofunctionalizedmembrane formed from these materials produces gel phase films at 25° C.where most experiments will be performed. If low p-gp activity is stillobserved, activity at higher temperatures (i.e. reduced microviscosity)or in the presence of mixed bolalipid-conventional lipid films (i.e.specific lipid requirement) can be evaluated.

Example IV Connection of the Biofunctionalized Membrane to the HydrogelMatrix

Hydrophilic polymers can be employed within the aqueous compartmenthousing the membrane film to connect the hydrogel support matrix to themembrane film to provide greater mechanical strength. The molecularentities used to chemically or physically connect the biofunctionalizedmembrane to the hydrogel matrix are selected so as to optimize theseparation distance between the two structures. This is important sincethe ABC site of P-gp may protrude 50 Å or more beyond the membrane filminterface toward the nanoporous substrate surface. If this distance isnot carefully controlled, the activity of P-gp (within an otherwiseintact structure) may be lost. Hydrophilic polymers are selected thatoptimize polymer molecular weight, surface density and chemical orphysical attachment to achieve the best stabilized supported membrane.

A molecular based theoretical methodology that enables the calculationof the structural and thermodynamic properties of tethered polymerlayers (Szleifer et al., Adv. Chem. Phys. Vol. XCIV, Chap. 3, pgs.165-260 (1996); Szleifer, Science 2, 337-344 (1997); and Szleifer etal., Macromolecular Rapid Communications 21, 423448 (2000)) can be usedto aid in the selection process. The predictions of the theory have beenshown to be in excellent quantitative agreement with experimentalobservations of pressure-area isotherms of PEG tethered chains (Faure etal., Eur. Phys. J.B 3, 365-375 (1998)), the ability of PEG to reduceprotein adsorption on hydrophobic surfaces (McPherson et al., Langmuir14, 176-186 (1998); and Satulovsky et al., Proc. Nat. Acad. Sci. 97,9037-9041 (2000)) and the effect of PEG to stabilize liposomes (Szleiferet al., Proc. Nat. Acad. Sci. 95, 1032-1037 (1998)).

To showcase the different possible supports that one can design, and thepossible consequences of not having the proper polymer layer,theoretical calculations were performed on two different scenarios. Inthe first, the interaction between two surfaces wherein the polymers arechemically attached to both surfaces was examined. The model polymerscorresponded to PEG-2200, and two surface coverages were examined. Inboth cases, the interactions between the surfaces had pronounced minimathat slightly moved toward larger distances when the surface density ofpolymer was increased. Further, the curvature of the interaction wasmuch larger for larger surface coverage, implying that the membrane willhave smaller fluctuations at larger surface coverages.

The second case corresponds to PEG-1000 that is chemically bound to thematrix, but interacts with the lipid bilayer that forms the membranefilm through a charged functionalized end-group in the PEG chain. Theresults correspond to relatively high surface coverages of polymer andlow salt concentration.

The shape of the potential in the second case was very different thanfor the first case. First, the attractive interaction showed a constantvalue over a relatively large range of distances. Second, the repulsionsat short distance were very sharp relative to the other case. Thisinteraction may cause the biofunctionalized membrane to become highlydelocalized throughout the minimal interaction region. Further, thispotential is very sensitive to salt concentration, suggesting that itmay change as P-gp transport of molecules occurs, if care is not takento keep the concentration of charged species constant.

These two examples show that the proper choice of surface coverage,molecular weight and type of attachment (chemical or physical) of thepolymer to the surface is important for the proper design of thebiofunctionalized membrane. A further complication that needs to beavoided is that the concentration of polymer in the supporting region besmall enough so that the analytes can reach and interact with thesensing molecules.

Tethering of the hydrophilic polymers such as PEG to the supportstructure and the biofunctionalized membrane can be accomplished by, forexample, covalent bonds at both ends of the hydrophilic polymer, or by acombination of covalent linkages and electrostatic interactions.

Double covalently-tethered chains. This corresponds to the case in whichthe PEG molecules are chemically bonded to both the lipid and matrix.The optimal distance between the aggregate and the hydrogel matrix isexpected to be on the order of 100 Å. Polymer molecular weight and rangeof surface coverage is selected for which the minimum in thesurface-surface interaction term is within 5 Å from the optimaldistance. The choice of surface coverage is preferably the minimalamount necessary to have interaction strength around the optimaldistance yet still enable the necessary freedom of membrane fluctuation.

Covalent and electrostatically-tethered chains. This corresponds to thecase of a chemical link between the polymer and the hydrogel matrix, andan electrostatic interaction—through the functionalized free-end of thepolymer—with charged lipids in the membrane. This may be a preferredconfiguration if covalent binding of the polymers to both interfacesresults in very restricted lateral motion of the lipids and protein onthe membrane. The stability of the electrostatic interactions needs tobe examined with respect to changes in salt concentration that may arisefrom the transport of drug candidates through the membrane.

Example V Microfabrication

The reaction chambers are fabricated using standard bulk micromachiningtechniques. Specifically, (100) silicon wafers with a boron etch stopembedded under 10 μm of silicon are acquired from a commercial source.The wafer is patterned using standard spin coating, exposure, anddevelopment protocols, and an anisotropic through etch will be used toform the reaction chambers. The optimum thickness of the silicon andetch stop conditions are determined. The silicon reaction chamber isbonded to commercially available nanoporous membranes that areapproximately 100 μm thick. After the bonding has been established thewafer supporting the reaction chambers are removed by back etching.Anodic etching protocols that produce nanoporous membranes that are bout10 μm thick are utilized.

Example VI Characterization of the Biofunctionalized Membrane

Signal Detection Techniques. Drug transport by P-gp requires ATPhydrolysis. Our sensor architecture utilizes ATP hydrolysis as the basisfor signal generation. This design generates a predictable response as aresult of drug-stimulated ATPase activity at 0.3-1.4 mM ATP levels.Impedance spectroscopy (IS) is well-suited for monitoring millimolarlevel changes in ionic conductance, therefore, this detection scheme ispreferred over those based on efflux of drugs whose physicochemicalproperties differ widely. Initially, IS of unbuffered ATP solutions inthe trapped aqueous layer is used to establish detection limits as afunction of P-gp loading density on the electrode surface.

If high background signal levels are observed due to basal, unstimulatedATPase activity that may occur prior to chemosensitizer activation,background can be suppressed by using materials with solid-gel meltingtransitions in the 10-15° C. range. Solidification of these films at 5°C. restricts the P-gp conformational freedom required for pumping andATPase activity. Heating above Tm before IS analysis restores ATPasecapacity. If ADP inhibition of ATPase activity limits the sensitivityachievable via IS technique, amperometric or pH detection can be adoptedas an alternative.

Atomic Force Microscopy. During the past decade, the atomic forcemicroscope (AFM) has become a key technique for the characterization ofsupported lipid films. The unique capabilities of the AFM include: (i)ability to probe, in real time and in aqueous environment, the surfacestructure of lipid films; (ii) ability to directly measure physicalproperties (i.e., thickness, surface forces, and elastic modulus) withnanometer scale spatial resolution. AFM can be used to characterize thelipid films at the solid liquid interface (Dufrene et al., Biomembranes1509, 14-41 (2000); Schneider et al., Biophysical Journal 79, 1107-1118(2000); and Dufrene et al., Faraday Discussions 111, 79-94 (1999)) toguide the work on forming orientating P-gp layers and designing novelmembranes to support these proteins. Specifically, the high spatialimaging capabilities of the microscope can be used to visualize thedistribution of P-gp at the solid liquid interface and characterize thequality of the lipid bilayer formed around the protein. Surface forcesand mechanical measurements can be used to characterize mechanisms ofadsorption and stability of the film.

Ellipsometry, IRRAS and Total Internal Reflection Microscopy. Films canbe characterized with ellipsometry, which probes films by measuring thereflectance and change of phase of polarized light beams. For bettersensitivity, multiple incident angles and wavelengths will be used(Walsh et al., J. Colloid Interf. Sci. 233, 295-305 (2001); and Walsh etal., Thin Solid Films 347, 167-177 (1999)). The measured ellipsometricparameters (angles Δ and ψ) can be used in solving inverse problems fordetermining thickness d, refractive index, and anisotropy of transparentor absorbing films for 0.01 μm to about 1 μm. For thinner films, only acombination of thickness and refractive index can be determined, or thesurface density of surfactants or lipid membranes.

In addition, infrared spectroscopy can be used to characterize surfacedensities of particular molecules, and their average orientation andchain conformation. Primarily, the IRRAS (reflection absorption)spectroscopy will be used, for layers as thin as 1-2 nm and as thick as0.1 μm.

Total internal reflection (TIR) fluorescence microscopy combines thestandard features of an epifluorescence microscope with uniqueevanescent wave excitation optics. At the interface between a quartzprism through which a laser beam comes in at a large incidence angle andtotally reflected, and a thin sample layer over which theelectromagnetic field does not abruptly drop to zero but decaysexponentially. This surface electromagnetic field, called the“evanescent wave,” can selectively excite fluorescent molecules in thethin liquid layer within 100 nm of the interface. The unique advantageof this design of optical path over others is that the background noisedue to the illuminating beam is completely rejected by total internalreflection (TIR). The technique has been shown ideally suited fordetecting single fluorescent molecules such as a molecular motor movingalong some fixed track or a labeled ligand attached to a largefunctional complex. The TIR microscopy is well suited for characterizingasymmetric membrane as the transfer of labeled protein across the lipidbi-layer is expected to cause a detectable change of signal. Thetechnique is also well suited for detecting slow migration of a labeledprotein probe within a 2-dimensional membrane fluid.

A second optical technique that is useful for structuralcharacterization of asymmetric membranes is by measurement of localbirefringence. Lipid membranes are know to be liquid crystallinematerials, which interact with polarized light in ways that can bemeasured using a special polarization optics setup (Oldenbourg et al.,J. of Microscopy 180, 140-147 (1995)). The sensitivity of the existingsetup is better than a nanometer of the retardance value, with spatialresolution of a few micrometers using a 40× objective. It is useful fordetecting changes within individual micro-arrays of the compositemembrane, such as the layer thickness and the local molecularorientation.

A third technique is a setup for microelectronic measurements. Ourapproach is similar to that developed to count polymers moving through asingle ion channel (Bezrukov et al., Nature 370, 279-281 (1994)), exceptthat the membrane will be laid flat on a homemade holder, with twoelectrodes above and below the membrane surface submerged in the aqueousmedium. The measured electric conductivity signal through the compositemembrane provides an independent report of membrane permeability tosmall ions of interest. In addition, since the whole device can beplaced on a microscope stage, the conductivity measurements can becombined with optical microscopy, including direct visualization ofprotein probes in the same membrane array using fluorescent tags.

The complete disclosures of all patents, patent applications includingprovisional patent applications, and publications, and electronicallyavailable material (e.g., GenBank amino acid and nucleotide sequencesubmissions) cited herein are incorporated by reference. The foregoingdetailed description and examples have been provided for clarity ofunderstanding only. No unnecessary limitations are to be understoodtherefrom. The invention is not limited to the exact details shown anddescribed; many variations will be apparent to one skilled in the artand are intended to be included within the invention defined by theclaims.

1. A bioanalytical device comprising: (a) a membrane film including monolayer membrane components selected from the group consisting of bolalipids, bolaamphiphiles, and amphiphilic tri-block copolymers, said membrane having an outer surface and an inner surface; (b) a support substrate in fluid communication with the inner surface of the membrane film and defining an aqueous compartment between the membrane film and the support substrate; (c) a transducer orienting layer overlapping said substrate and including orienting moieties effective for orienting a protein transducer; and (d) a protein transducer within said membrane film and directionally oriented therewith by interaction with said orienting moieties of said transducer orienting layer.
 2. (The bioanalytical device of claim 1, wherein said interaction of said protein transducer and said transducer orienting layer is reversible.
 3. The bioanalytical device of claim 1, wherein said interaction of said protein transducer and said transducer orienting layer is irreversible.
 4. The bioanalytical device of claim 1, wherein a single layer functions as said support substrate and said transducer orienting layer.
 5. The bioanalytical device of claim 1, wherein said transducer orienting layer includes polyethylene glycol.
 6. The bioanalytical device of claim 5, wherein said orienting moieties include α-(α-lysinenitrilotriacetic acid (NTA).
 7. The bioanalytical device of claim 1, wherein said membrane component includes a bolalipid.
 8. The bioanalytical device of claim 1, wherein said membrane component includes a bolaamphiphile.
 9. The bioanalytical device of claim 1, wherein said membrane component includes an amphiphilic tri-block copolymer.
 10. The bioanalytical device of claim 1, wherein said orienting moieties include affinity tags selected from the group consisting of His-, Myc-, FLAG-, and HA-.
 11. The bioanalytical device of claim 10, wherein said affinity tag is a polyhistidine-Ni²⁺-NTA moiety.
 12. The bioanalytical device of claim 1 wherein the transducer orienting layer comprises a detection moiety for detecting the activity of the protein transducer.
 13. The bioanalytical device of claim 1 wherein said protein transducer is in a perpetual “on” state allowing an analyte constant passage through said membrane film.
 14. A method for analyzing an analyte comprising: contacting the analyte with the outer surface of the membrane film of a device as in claim 1, such that the analyte passes into or across the membrane film; and detecting the activity of the protein transducer, wherein activity of the protein transducer is indicative of the efflux of the analyte from the outer surface of the membrane film.
 15. The method of claim 14, wherein the analyte is a candidate drug.
 16. A bioanalytical device comprising: (a) a monolayer membrane film having an outer surface and an inner surface; (b) a support substrate in fluid communication with the inner surface of the membrane film and defining an aqueous compartment between the membrane film and the support substrate. (c) a transducer orienting layer overlapping said substrate and including orienting moieties effective for orienting a protein transducer; and (d) a protein transducer within said membrane film, wherein said transducer is directionally oriented therewith by interaction with said orienting moieties of said transducer orienting layer and in a perpetual “on” state allowing an analyte constant passage through said membrane film.
 17. The bioanalytical device of claim 16, wherein said membrane film includes a component selected from the group consisting of bolalipids, bolaamphiphiles, and amphiphilic tri-block copolymers.
 18. The bioanalytical device of claim 16 wherein the bioanalytical device is fabricated as a replaceable cartridge.
 19. A method for identifying an inhibitor of a protein transducer comprising: contacting the outer surface of the membrane film of a device as in claim 16 with: (a) an analyte capable of being actively transported by the protein transducer, wherein the protein transducer is a membrane-embedded protein transducer and (b) a candidate inhibitor of the protein transducer; and determining whether the active transport of the analyte by the membrane-embedded protein transducer is inhibited by the candidate compared to the active transport of the analyte by the membrane-embedded protein transducer in the absence of the candidate inhibitor.
 20. A method for making a bioanalytical device comprising: (a) applying a hydrogel precursor solution to the surface of a support substrate; (b) applying a gellating agent to the hydrogel precursor to cause gellation of the hydrogel precursors to yield a transducer orienting layer in physical contact with the surface of the support substrate, said transducer orienting layer including terminal orienting moieties for orienting a protein transducer; (c) applying a protein transducer and a membrane film material to said transducer orienting layer to yield a membrane film having said protein transducer oriented with said membrane film through said orienting moieties and an inner surface defining an aqueous compartment providing communication between said membrane film and said transducer orienting layer, said film material including at least one component selected from the group consisting of bolalipids, bolaamphiphiles, and amphiphilic tri-block copolymers.
 21. The method of claim 20, wherein the transducer orienting layer is integrated into the support substrate. 